(Lab period 10)
Cell fractionation and isolation of chloroplasts
The green color of the leaves, and sometimes the stems, of plants is due to the presence of the
green pigments chlorophyll
in the subcellular organelles called chloroplasts.
remainder of a green cell is typically colorless.
The green organelles lie free in the cytoplasm of the cell,
unattached to other cellular components such as the wall, the membranes, the nucleus, and the
When the cell wall is disrupted, the cell membrane breaks, and the subcellular
components are released as separate particles of various sizes and densities.
In this laboratory, the cells of spinach leaves will be disrupted, freeing the untethered organelles,
which can then be sorted out from each other by filtration and differential centrifugation as cell fractions.
Filtration will remove large debris (e.g., cell walls) and unbroken cells, providing a filtrate that contains
organelles (nuclei, chloroplasts, mitochondria, ribosomes), small membrane vesicles, and soluble
components; most of these will not be visible in the light microscope.
Low-speed centrifugation will
sediment remaining large bodies from the filtrate, and moderate-speed centrifugation will sediment
chloroplasts, leaving mitochondria and ribosomes and soluble components in the supernatant.
mitochondrial fraction could be collected by high-speed, and ribosomal and membrane vesicle fractions
by ultra-high-speed centrifugation.)
Repeated rounds of differential centrifugation can be used to further
purify the chloroplasts when highly purified preparations are required for experimentation, but one round
of low-then-moderate centrifugation will suffice for the purposes of this exercise.
In this laboratory, you will prepare a crude suspension of chloroplasts and determine several